Oxidation of NADH by plant mitochondria: Kinetics and effects of calcium ions

The kinetics of NADH oxidation by the outer membrane electron transport system of intact beetroot (Beta vulgaris L.) mitochondria were investigated. Very different values for V_max and the K_m for NADH were obtained when either antimycin A‐insensitive NADH‐cytochrome c activity (V_max= 31 ± 2.5 nmol cytochrome c (mg protein)⁻¹ min⁻¹; K_m= 3.1 ± 0.8 μM) or antimycin A‐insensitive NADH‐ferricyanide activity (V_max= 1.7 ± 0.7 μmol ferricyanide (mg protein)⁻¹ min⁻¹; K_m= 83 ± 20 μM) were measured. As ferricyanide is believed to accept electrons closer to the NADH binding site than cytochrome c, it was concluded that 83 ± 20 μM NADH represented a more accurate estimate of the binding affinity of the outer membrane dehydrogenase for NADH. The low K_m determined with NADH‐cytochrome c activity may be due to a limitation in electron flow through the components of the outer membrane electron transport chain. The K_m for NADH of the externally‐facing inner membrane NADH dehydrogenase of pea leaf (Pisum sativum L. cv. Massey Gem) mitochondria was 26.7 ± 4.3 μM when oxygen was the electron acceptor. At an NADH concentration at which the inner membrane dehydrogenase should predominate, the Ca²⁺ chelator, ethyleneglycol‐(β‐aminoethylether)‐N,N,‐tetraacetic acid (EGTA), inhibited the oxidation of NADH through to oxygen and to the ubiquinone‐10 analogues, duroquinone and ubiquinone‐1, but had no effect on the antimycin A‐insensitive ferricyanide reduction. It is concluded that the site of action of Ca²⁺ involves the interaction of the enzyme with ubiquinone and not with NADH.